<i>CBL-INTERACTING PROTEIN KINASE 9</i> regulates ammonium-dependent root growth downstream of <i>IDD10</i> in rice (<i>Oryza sativa</i>)

Abstract

Background and Aims INDETERMINATE DOMAIN 10 (IDD10) is a key transcription factor gene that activates the expression of a large number of NH4+-responsive genes including AMMONIUM TRANSPORTER 1;2 (AMT1;2). Primary root growth of rice (Oryza sativa) idd10 mutants is hypersensitive to NH4+. The involvement of CALCINEURIN B-LIKE INTERACTING PROTEIN KINASE (CIPK) genes in the action of IDD10 on NH4+-mediated root growth was investigated. Methods Quantitative reverse transcription-PCR was used to analyse NH4+- and IDD10-dependent expression of CIPK genes. IDD10-regulated CIPK target genes were identified using electrophoretic mobility shift assays, chromatin immunoprecipitation and transient transcription assays. Root growth rate, ammonium content and N-15 uptake of cipk mutants were measured to determine their sensitivity to NH4+ and to compare these phenotypes with those of idd10. The genetic relationship between CIPK9 OX and idd10 was investigated by crosses between the CIPK9 and IDD10 lines. Key Results AMT1;2 was overexpressed in idd10 to determine whether NH4+-hypersensitive root growth of idd10 resulted from limitations in NH4+ uptake or from low cellular levels of NH4+. High NH4+ levels in idd10/AMT1;2 OX did not rescue the root growth defect. Next, the involvement of CIPK genes in NH4+-dependent root growth and interactions between IDD10 and CIPK genes was investigated. Molecular analysis revealed that IDD10 directly activated transcription of CIPK9 and CIPK14. Expression of CIPK8, 9, 14/15 and 23 was sensitive to exogenous NH4+. Further studies revealed that cipk9 and idd10 had almost identical NH4+-sensitive root phenotypes, including low efficiency of (NH4+)-N-15 uptake. Analysis of plants containing both idd10 and CIPK9 OX showed that CIPK9 OX could rescue the NH4+-dependent root growth defects of idd10. Conclusions CIPK9 was involved in NH4+-dependent root growth and appeared to act downstream of IDD10. This information will be useful in future explorations of NH4+ signalling in plants.